Methods for modulating phototoxicity

ABSTRACT

The invention relates to methods for modulating photodamage via the use of collagen derived molecules which either enhance or inhibit damage caused by ultraviolet light.

RELATED APPLICATION

[0001] This application claims priority of provisional application60/374,033, filed Apr. 19, 2002. The entire disclosure of thisapplication is incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to molecules and compositions which areuseful as modulators of skin cell phototoxicity. Specifically,“modulators” as used herein refers to materials which can eitheraccelerate or retard the damage of cells, such as skin cells, caused byexposure to light.

BACKGROUND AND PRIOR ART

[0003] It is well known that light, UVA light in particular, is damagingto skin cells. Phototoxic cell damage occurs via the reaction of lightwith certain compounds that are endogenous to skin. The mechanism bywhich the photodamage occurs is well understood, and can be described,briefly, as follows. The molecules involved, which may be referred to assensitizers or even accelerators of skin damage, react with light and,in the presence of oxygen, result in the formation of “reactive oxygenspecies”, or “ROS.” It is these molecules, i.e., ROS molecules, whichare involved in pathways leading to cell damage, includingcarcinogenesis and photoaging, but not being limited to these phenomena.More details of this phenomenon may be found in Wondrak, et al. J.Invest. Dermatol 119:489-498 (2002), the entirety of which isincorporated by reference.

[0004] The fact that molecules endogenous to, e.g., the skin, areinvolved in accelerated phototoxicity suggests targeted therapy. Toelaborate, if a compound is essentially inert in the absence of lightbut is involved in cellular destruction upon contact with light, thensuch compounds could be used in situations where targeted cell death isdesired. Such situations include, but are not limited to, psoriasis,acne, premalignant and malignant hyperproliferative disorders such asactinic keratosis, and other conditions well known to the art.

[0005] Conversely, the existence of the photoactivable moleculessuggests the existence of molecules which act to quench or to inhibitthe effect of light on cells. Such quenchers or inhibitors can be usedin situations where the harmful effects of light need to be reversed,and/or inhibited. Such quenchers or inhibitors may be usedprophylactically, as well as therapeutically.

[0006] Hence, the modulation of phototoxicity on cells is the focus ofthe invention described herein, as will be seen in the disclosure whichfollows. Modulators, as used herein, refers to molecules which may bederived from skin components, collagen in particular.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 sets forth the structures of various molecules employed inthe examples.

[0008]FIG. 2 depicts results showing that molecules with a 3hydroxypyridine central structure were efficacious in provokinginhibition of cell proliferation, when exposed to light.

[0009]FIG. 3 compares the results obtained for the 3-hydroxpyridinecompounds of FIG. 1, on HaCaT cells, which are keratinocytes, in thepresence of light.

[0010]FIG. 4 depicts result obtained on malignant melanoma, using theN-ethyl derivative described herein.

[0011]FIG. 5 parallels FIG. 4, but the cells used were breast cancercells.

[0012]FIG. 6 presents, via FACS (flow cytometric) analysis, evidencethat the N-ethyl derivative pushes cells into apoptosis.

[0013]FIG. 7 presents schematically the synthesis of BSA-B6 complexes.

[0014]FIG. 8 shows that the complexes of FIG. 7 were effective ininhibiting cell proliferation.

[0015]FIG. 9 is a proposed mechanism for the quenching of photoactivatedmolecules by energy transfer (“ET” in the figure). “S*” is the fullyexcited sensitizer, while “S” is the sensitizer.

[0016]FIG. 10 sets forth structures of proline derivatives tested asquenchers.

[0017]FIG. 11 depicts results from assays designed to show the efficacyof the proline derivatives in quenching photosensitized, DNA damage.

[0018]FIG. 12 shows how it was determined that compounds describedherein quenched singlet oxygen.

[0019]FIG. 13 shows the protective effect of various quencher moleculeswhich inhibit skin cell photodamage.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0020] Components of the endogenous skin component, to collagen werebelieved to be most likely the causative agents of phototoxic damageand/or inhibition thereof. Hence, such molecules were investigated.

[0021] Pyridinoline is an amino acid involved in cross linking collagenmolecules, and it was investigated for its role in phototoxicity. Thestructure of pyridinoline is well known, and is depicted in FIG. 1,together with the structure of other molecules described herein.

[0022] A series of experiments were carried out on HaCaT keratinocytes,and human CF3 fibroblasts. In these experiments, cell samples werecontacted with 500 μM of pyridinoline, 500 μM of desmosine, which is anelastin constituent with a structure related to pyridinoline, or 500 μMof vitamin B6 (pyridoxine). Controls received no added compound. In oneset of experiments, the cells received no external light source. Inanother set of experiments, they received UVA light, at 3.3 J/cm². TheCF3 fibroblasts received solar simulated light, or “SSL,” which combines2.3 J/cm² of UVA light, and 0.12 J/cm² of UVB light. Results are shown,in FIG. 2, in terms of percent of cell proliferation relative to acontrol (no added compound, no light). The measurements were taken 3days after stimulation.

[0023] The results indicated that pyridinoline had an antiproliferativeeffect, but only in the presence of light. Vitamin B6 showeddramatically more efficacy in inhibiting the cellular proliferation.

[0024] A second set of experiments were carried out in which catalase,which is a peroxide scavenger, was added, at 400 μ/ml. The catalase hadabsolutely no effect on vitamin B6 sensitization, suggesting that amechanism other than peroxide formation was involved for this molecule.

[0025] Based upon these results, structures of the compounds werecompared to determine if a common structural feature of the molecules,or a “pharmacophore” could be identified. It was noted that both vitaminB6 and pyridinoline share a 3-hydroxypyridine central structure, whichin turn suggested the next series of experiments.

EXAMPLE 2

[0026] A group of hydroxypyridine derivatives were studied, inexperiments paralleling those described supra. In brief, 2, 3, and4-hydroxypyridine were tested, as was N-ethyl-3-hydroxypyridine. Allstructures are set forth in FIG. 1.

[0027] HaCaT cells, as described supra, were tested in a proliferationinhibition assay. Cell samples received equal amounts of one of the 4compounds listed supra and were contacted with or without either solarsimulated light (“SSL”) as described, supra, or UVA light alone, also asdescribed supra.

[0028] The results are depicted in FIG. 3.

[0029] As compared to controls, 3-hydroxypyridine had an inhibitoryeffect, where the N-ethyl derivative had a killing effect. The N-ethylderivative also functioned in the presence of both UVA and UVB light,whereas 3-hydroxypyridine functioned in the presence of UVB light as acell proliferation inhibitor.

EXAMPLE 3

[0030] The experiments described supra were followed by additionalexperiments using malignant melanoma cells (G-361 cells ), and malignantbreast cancer cells (MCF-7). In these experiments, the N-ethylderivative described supra was tested as described, at varyingconcentrations, with UVA light at 9.9 J/cm². Viability was measured twodays following the treatment. As controls, experiments were run usingonly the N-ethyl derivative, and only the UVA light.

[0031] The results, shown in FIGS. 4 (G-361 cells) and 5 (MCF7 cells),show that the combination led to pronounced cytotoxicity.

[0032] When the G-361 cells were analyzed via FACS, the data showed thatthe cells were driven into programmed cell death, i.e., apoptosis. Thisis seen in FIG. 6, where staining with the apoptic markers annexin V andpropidium iodide showed a sharp increase in stained cells when theN-ethyl derivative was used, especially with UVA at a concentration of3.3 J/m².

EXAMPLE 4

[0033] The effective materials of the invention are small molecules.While small molecules are useful, it is sometimes desirable to complexsuch molecules to larger molecules, such as proteins. This facilitatestargeting of the small molecule if it is complexed with, e.g., anantibody specific for a particular marker on cells, a ligand for aparticular receptor, a nuclear associated protein and so forth.

[0034] To test the feasibility of this approach, vitamin B6 moleculeswere coupled to bovine serum albumin. In brief, a lysine side chain ofthe bovine serum albumin (BSA) molecule was covalently modified byreacting 350 mg of it with vitamin B6, which is a pyridoxal aldehyde (64mg), to form a Schiff base. In turn, the Schiff base was reduced withNaCNBH₃ (58 mg) in 1.5 ml of 0.25 M phosphate buffer (pH 7.4), overnightat 37° C., and dialyzed extensively (48 hours, 4° C.). The resultingBSA-B6 adduct was characterized by mass spectrometry and fluorescencespectroscopy. The protein was then lyophilized, and used in the exampleswhich follow. FIG. 7 depicts the synthesis. The spectroscopy workindicated that, on average, each BSA molecule was complexed to 5-6pyroxidal molecules.

EXAMPLE 5

[0035] The antiproliferative effect of the complexes described inexample 4 were tested, by adding either nothing (control), BSA, or theBSA-B6 complexes, and treating or not treating samples of HaCaTkeratinocytes with SSL. The BSA and BSA-B6 were added at 10 mg/ml, andthe SSL was 2.3 J/cm² of UVA plus 0.12 J/cm² UVB. Three days aftertreatment, proliferation was measured using a Coulter counter, andstandard methods.

[0036] It will be seen from the results shown in FIG. 8 that the BSA-B6complexes were very effective in inhibiting the proliferation of thekeratinocytes.

EXAMPLE 6

[0037] The data set forth in examples 1-5, supra, deal with moleculeswhich enhance cellular destruction. Such is not always desirable,however, and in this example and those which follow, experiments are setforth which describe molecules which inhibit this process. Thesemolecules will be referred to as quenchers of photoexcited states, or“QPES” hereafter. Such compounds are characterized by an ability toinactivate the photoexcited state of a molecule which would then provokethe type of cell death described supra.

[0038] The proposed mechanism by which these molecules function is setforth in FIG. 9, although it is to be noted that applicants do not wishto be bound by this proposed mechanism. In brief, UV irradiation of amolecule leads to excitation of electrons (excited states, as “S*” inthe figure), together with formation of excited singlets, and, afterintersystem crossing (ISC), triplet states. These are key intermediatesin the photodamage of cells. QPES compounds nullify this effect byaccepting the excitation energy of the compounds associated withphotodamage by energy transfer (“ET”), neutralizing phototoxicintermediates which relax back to ground state, dissipating the energyvia harmless vibrational energy, or heat. The QPES compounds themselvesare not depleted in this process, and neutralize multiple photodamagingmolecules.

EXAMPLE 7

[0039] It is well known that ΦX174 plasmid is cleaved only by combinedaction of irradiation from solar simulated light, and AGE-pigmentenriched protein, which acts as a UV sensitizer. AGE-BSA (“advancedglycation end product” modified bovine serum albumin), is a model foraccumulation of endogenous skin sensitizers of the type described supra.Details of the assay showing this will be found in Wondrak, et al.,Photochem. Photobiol. Sci. 1:355-363 (2002) incorporated by reference inits entirety. This assay was used in this example.

[0040] Plasmid cleavage was visualized by running samples on 1% agarosegels, and damage, i.e., formation of relaxed, open circular forms fromclosed circular forms (undamaged) were quantified via densitometry,which permitted assessment of the protective effect of a compound.

[0041] AGE-cleavage proceeds in the absence of oxygen, and cannot besuppressed fully via antioxidants. As such, if a compound suppressesplasmid cleavage it cannot be simply considered an antioxidant. Incontrast, inhibition via quenching of the excited state, as describedsupra, must be presumed.

[0042] The results are presented in the Table which follows. CytotoxicNaN₃, which is known as a quencher of photoexcited states, waseffective, as were thiol compounds including glutathione (“GSH”),D-penicillamine, and N-acetyl-L-cysteine (“NAC”).

[0043] This assay proves the principle discussed in example 7. TABLE 1Suppression of AGE-Sensitized DNA cleavage Compound % Inhibition (±SD)Catalase [400 μ/mL] 57.5 ± 4.7 SOD [300 μ/mL] 48.0 ± 1.1 Marmitol [20mM] 47.5 ± 2.5 L-Histidine [20 mM] 43.7 ± 0.4 NaN₃ [20 mM] 94.0 ± 0.5DABCO [20 mM] 32.9 ± 1.1 DFO [1 mM] 51.3 ± 1.0 D-Penicillamine [20 mM]95.7 ± 0.7 NAC [20 mM] 99.4 ± 0.9 GSH [20 mM] 100.0 ± 0.0 

[0044] Using this assay, a series of proline related compounds andproline were tested. The structures of the compounds are set forth atFIG. 10. FIG. 11, sets forth the results of these experiments. The testcompound was added, at 100 mM, and the plasmids were contacted with one,both, or neither of UVA light (2.3 J/cm²), and AGE-BSA (10 mg/ml).

[0045] L-Pro-OCH₃ was the most potent of the quenchers, but all wereeffective.

EXAMPLE 8

[0046] As was discussed, supra, photoexcited oxygen, i.e., singletoxygen, or “¹O₂”, which is a spin paired homologue of ground state,triplet oxygen or “³O₂,” is the most important excited state moleculeknown to be in involved in phototoxicity. The involvement of singletoxygen in photooxidative damage to, inter alia, cellular DNA, membranelipids, and structural proteins like keratin and collagen, has been welldocumented. An assay was developed to determine the efficacy ofcompounds, such as proline and the proline derivatives described supra,in quenching singlet oxygen. See Lion, et al., Nature 263:442-443(1976), incorporated by reference in its entirety.

[0047] It is well known that toluidine blue (“TB”), upon irradiation,generates ¹O₂. In a known methodology, the ¹O₂ is trapped via 2, 2, 6,6, tetramethylpiperidine, or “TEMP.” A stable free radical, i.e., 2, 2,6, 6 tetramethylpiperdine-1-oxyl, or “TEMPO” forms, which can then bemeasured as a determination of ¹O₂ generation. Key to this assay is thefact that other reactive oxygen species do not react with TEMP toproduce TEMPO, thus ensuring that the assay is specific for formation of¹O₂.

[0048] TEMP, TB, and one of the molecules described supra were combined,and irradiated with visible light for 5 minutes, receiving a total doseof 36 J/cm². To elaborate, 100 μM TB, 7 mM TEMP, and 20 mM test compoundwere combined, in phosphate buffered saline, in a quarz capillary tubeof 100 μl volume (1.5×90 mm), TEMPO free radical signal was measured viaelectron paramagnetic resonance using a commercially availableapparatus. Controls were also run, to determine signal in the absence oftest compound but in the presence of light, and the standard TEMPOresonance signal.

[0049] The results, presented in FIG. 12, panels A-G, provide data asfollows. Panel A shows the complete singlet oxygen generating systemwith production of the TEMPO signal. Panel B shows the spin signal of acommercial TEMPO reference. Panel C demonstrates that the effectsobserved in panels D-G were not caused by direct reaction of testcompounds with TEMPO. Panels D & G demonstrate singlet oxygen quenchingby proline derivatives and prove that derivatization of the carboxygroup of proline or 4-hydroxylation does not impact quenching activity.Further, it shows that all of the compounds were effective quenchers.

[0050] In order to show that the quenching molecules were physicalquenchers and were not consumed in the reaction, amino acid content of amixture of TB and L-Pro in PBS was measured before and after a prolongedexposure to visible light (36 J/cm²), using standard reversed phase HPLCmethods for amino acid analysis. No changes were observed in the peak,retention time, or AUC values, indicating chemical inertness toward the¹O₂, providing evidence of physical quenching, rather than reactantconsumption.

EXAMPLE 9

[0051] These experiments were designed to determine if the compoundsproven to be quenchers were efficacious in protecting skin cells.

[0052] Cultured skin fibroblasts (CF3 cells) were exposed tophotooxidative stress from ¹O₂ which had formed, in situ, by dyesensitization.

[0053] In brief, 50,000 fibroblasts were seeded in a 35 mm culture dish,and then treated one day later with visible light (90 seconds exposure,providing 10.8 J/cm²) in the presence or absence of TB (3.3 μM in Hanksbuffered salt solution). Five minutes after treatment, cells werewashed, with phosphate buffered saline. Test compound (10 mM) waspresent or absent in the culture during the radiation. After 3 days ofcultivation, cells were harvested via trypsinization, and counted usinga Coulter counter.

[0054] The results indicated that cell proliferation was highlysuppressed (70%) by the combination of TB and light, but either of thesealone did not do so. The compounds L-Pro-OCH₃ and 4-OH-L-Pro-OCH₃ showeda very clear protective effect, as is shown in FIG. 13 and quantified inthe table which follows: Cell Protection against Sensitized Photodamage[% ± SD, n = 3] QPES-compound: L-Pro 6.2 ± 5.4 L-Pro-OCH₃ 83.8 ± 10.94-OH-L-Pro 7.3 ± 2.5 4-OH-L-Pro-OCH₃ 71.0 ± 13.4

[0055] Protection was determined via the formula:${{protection}\quad (\%)} = {\left\lbrack \frac{\begin{matrix}{\% \quad {proliferation}\quad \left( {\left( {{TB} + {Light} + {QPES}} \right) -} \right.} \\{\% \quad {proliferation}\quad \left( {{TB} + {Light}} \right)}\end{matrix}}{\begin{matrix}{{\% \quad {proliferation}\quad ({Control})} -} \\{\% \quad {proliferation}\quad \left( {{TB} + {Light}} \right)}\end{matrix}} \right\rbrack \times 100\%}$

[0056] The more active compounds in these experiments were the estercompounds. As is shown in FIG. 10, when the log P values of thesecompounds were determined, using methods well known in the art, theesters had significantly higher log P values than the non-esterifiedcompounds. As the higher log P values are indicative of greaterlipophilicity, it may be the case that their superior efficacy is due tomaintaining longer residence time in skin and interaction with cellmembranes. Proline ester derivatives with a log P value of from about−1.00 to about +8.00 are expected to be especially useful.

EXAMPLE 11

[0057] The compound 4-OH-L-proline methyl ester, described supra, isbelieved to be a new compound, representative of a new group ofcompounds. The synthesis, of the compound and guidelines for synthesisof other members of this family are now set forth.

[0058] 4-hydroxy-L-proline was reacted with di-tert-butyl dicarbonate,in 1N NaOH at 5° C. for 30 minutes, followed by stirring, at roomtemperature, for an additional 3.5 hours.

[0059] The resulting protected 4-hydroxy proline was then reacted withdimethylformamide with potassium carbonate and methyl iodide, at 0° C.,for 30 minutes, followed by stirring for an hour, at room temperature,to produce the methyl ester.

[0060] Deprotection of tert-butyl dicarbonate was then carried out usingtrifluoroacetic acid, and standard methods.

[0061] The key component in this reaction is methyl iodide. By varyingthe alkyl halide, esters containing alkyl groups with from 1-30,preferably 1-26 carbon atoms, can be obtained.

[0062] The foregoing examples set forth various aspects of theinvention, which relate to methods for modulating photodamage, byadministering at least one substance which modulates the effect ofultraviolet light on a subject. “Modulates” as used herein, refersgenerically to the ability to either increase the rate of photodamage,which is useful in situations where cellular proliferation needs to bearrested or to decrease the rate of damage. Examples of the formercategory include, e.g., psoriasis, acne, premalignant and malignanthyperproliferative disorders such as actinic keratosis, melanoma,non-melanoma skin cancer, breast cancer, and other cancers, as well asother conditions which will be known to the skilled artisan as involvingthe need to reduce cellular proliferation.

[0063] It is preferred that increase in photodamage, leading to adecrease in cellular proliferation, be accomplished via the use of atleast one compound which has, as its “pharmacaphore” structure, a3-hydroxypyridine ring, and is derived from a skin component, such ascollagen. Exemplary of such compounds are 3-hydroxypyridine itself,vitamin B6, and most preferably, N-alkyl 3-hydroxypyridiniumderivatives, such as salts, wherein the alkyl chain of the derivativecomprises at least 2 and as many as 20 carbon atoms, in a preferablystraight (but optimally branched) chain, which may or may not besubstituted. More preferably, the alkyl group contains 2-10 carbon atomsin a straight chain, and most preferably, 2-5 carbon atoms. As was shownvia the data set forth, supra, the N-ethyl derivative is especiallypreferred. Longer N-alkyl derivatives, which enhance the length of timethe compound resides in the skin may be preferred in particularsituations, such as, but not being limited to, situations where topicaldelivery of the compound is desired.

[0064] These molecules may be combined with, e.g., standardpharmaceutical ingredients and carriers, such as those employed increams, lotions, shampoos, sprays, patches, or any of the knownpharmacological delivery systems that are used for administeringtherapeutic agents to skin.

[0065] Where targeted therapy is especially desirable, the activeingredients may be attached to a second molecule which may be larger, toimprove targeted delivery. Such larger molecules include, e.g.,antibodies, ligands for receptors, hormones, and other molecules whicheither target and/or are taken up selectively by cells.

[0066] A further feature of the invention is the quenching feature ofphotodamage. As was shown, supra, proline and derivatives thereof areeffective quenchers of photodamage. Especially efficacious are alkylesters of proline, such as L-Pro-OCH₃ and 4-OH-L-proline; however, othercompounds are useful, as has been shown. The modes of application areprecisely as is described for the enhancers discussed, supra.

[0067] Other features of the invention will be clear to the skilledartisan, and need not be reiterated herein.

We claim:
 1. A method for modulating photodamage in a subject in needthereof, comprising applying to skin of said subject an amount of aphotodamage modulator sufficient to modulate photodamage caused byultraviolet light.
 2. The method of claim 1, wherein said modulatorenhances photodamage.
 3. The method of claim 2, wherein said modulatoris collagen derived.
 4. The method of claim 2, wherein said modulatorhas a structure which consists of a 3-hydroxypyridine pharmacophore. 5.The method of claim 4, wherein said modulator is anN-alkyl-3-hydroxypyridine or salt thereof.
 6. The method of claim 5,wherein said N-alkyl-3-hydroxypyridine or salt thereof comprises analkyl moiety of 2-20 carbon atoms.
 7. The method of claim 6, whereinsaid alkyl moiety is an ethyl group.
 8. The method of claim 2, whereinsaid modulator is connected to a carrier molecule.
 9. The method ofclaim 2, wherein said modulator is vitamin B6, pyridinoline, or3-hydroxypyridine.
 10. The method of claim 1, wherein said modulatorinhibits photodamage.
 11. The method of claim 10, wherein said modulatoris proline, 4-hydroxyproline or an alkyl ester thereof.
 12. The methodof claim 11, wherein said proline alkyl ester comprises an alkyl moietyof 1-24 carbon atoms.
 13. The method of claim 12, wherein said prolinealkyl ester is L-proline-OCH₃ or 4-OH-L-Pro-OCH₃.
 14. The method ofclaim 1, comprising applying said modulator in the form of a cream,lotion, shampoo, spray or patch.
 15. A 4-hydroxy-L-proline alkyl estercompound having an alkyl chain which contains from 1 to 24 carbon atoms.16. The compound of claim 15, which has a log P from about −1.00 toabout +8.00.
 17. The compound of claim 15, wherein said alkyl chaincontains 1-10 carbon atoms.
 18. The compound of claim 15, wherein saidalkyl chain contains from 16 to 22 carbon atoms.
 19. The compound ofclaim 15, wherein said alkyl chain contains from 11-24 carbon atoms. 20.The compound of claim 15, designated 4-hydroxy-L-proline methyl ester.